Finding evolutionary relations beyond superfamilies: fold-based superfamilies

Superfamily classifications are based variably on similarity of sequences, global folds, local structures, or functions. We have examined the possibility of defining superfamilies purely from the viewpoint of the global fold/function relationship. For this purpose, we first classified protein domains according to the beta-sheet topology. We then introduced the concept of kinship relations among the classified beta-sheet topology by assuming that the major elementary event leading to creation of a new beta-sheet topology is either an addition or deletion of one beta-strand at the edge of an existing beta-sheet during the molecular evolution. Based on this kinship relation, a network of protein domains was constructed so that the distance between a pair of domains represents the number of evolutionary events that lead one from the other domain. We then mapped on it all known domains with a specific core chemical function (here taken, as an example, that involving ATP or its analogs). Careful analyses revealed that the domains are found distributed on the network as >20 mutually disjointed clusters. The proteins in each cluster are defined to form a fold-based superfamily. The results indicate that >20 ATP-binding protein superfamilies have been invented independently in the process of molecular evolution, and the conservative evolutionary diffusion of global folds and functions is the origin of the relationship between them.     

Roles of hemoglobin Allostery in hypoxia-induced metabolic alterations in erythrocytes: simulation and its verification by metabolome analysis

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [(13)C]glucose to [(13)C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O(2)-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O(2) from the cells.     

Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase

The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The KS-catalyzed reaction involves two discrete steps consisting of formation of an acyl-enzyme intermediate generated from the incoming acylthioester substrate and an active site cysteine residue, and the conversion of this intermediate to the beta-ketoacyl-acyl carrier protein product by a decarboxylative condensation with a paired methylmalonyl-SACP. We have determined the rate constants for the individual biochemical steps by a combination of protein acylation and transthioesterification experiments. The first-order rate constant (k(2)) for formation of the acyl-enzyme intermediate from [1-(14)C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 +/- 2.6 min(-)(1), with a dissociation constant (K(S)) of 3.5 +/- 2.8 mM. The acyl-enzyme adduct was formed at a near-stoichiometric ratio of approximately 0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-(14)C-acetyl]cysteamine gave a k(exch) of 0.15 +/- 0.06 min(-)(1), with a K(m) for HSNAC of 5.7 +/- 4.9 mM and a K(m) for 2 of 5.3 +/- 0.9 mM. Under the conditions that were used, k(exch) was equal to k(-)(2), the first-order rate constant for reversal of the acyl-enzyme-forming reaction. Since the rate of the decarboxylative condensation is much greater that the rate of reversion to the starting material (k(3) > k(-)(2)), formation of the acyl-enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (k(cat)/K(m)) is solely a reflection of the intrinsic substrate specificity of the KS-catalyzed acyl-enzyme-forming reaction. These findings were also extended to a panel of diketide- and triketide-SNAC analogues, revealing that some substrate analogues that are not converted to product by DEBS module 2 form dead-end acyl-enzyme intermediates.     

2D7 diabody bound to the alpha2 domain of HLA class I efficiently induces caspase-independent cell death against malignant and activated lymphoid cells

A mouse monoclonal antibody (2D7 mAb), which specifically bound to the alpha2 domain of HLA class I, rapidly induces cell aggregation accompanied by weak cytotoxicity against ARH-77 cells, suggesting that 2D7 mAb had a potential for agonist antibody. In order to enhance this cytotoxicity, 2D7 mAb was engineered to be a small bivalent antibody fragment, 2D7 diabody. The resultant 2D7 diabody showed a strong cytotoxicity against ARH-77 cells. As a notable characteristic feature, the lethal effect of 2D7 diabody was quite rapid, mediated by a caspase-independent death pathway. Furthermore, 2D7 diabody also showed cytotoxicity against several leukemia and lymphoma cell lines, and mitogen-activated peripheral blood mononuclear cells (PBMC), but not for normal resting PBMC and adherent cell lines such as HUVEC. These results suggest that 2D7 diabody could be expected as a novel therapeutic antibody for hematological malignancies as well as inflammatory diseases.     

Theonellamide A,a marine-sponge-derived bicyclic peptide,binds to cholesterol in aqueous DMSO: Solution NMR-based analysis of peptide-sterol interactions using hydroxylated sterol

Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. The inclusion of cholesterol (Chol) or ergosterol in the phosphatidylcholine membrane is known to significantly enhance the membrane affinity for theonellamide A (TNM-A). We have previously revealed that TNM-A stays in a monomeric form in dimethylsulfoxide (DMSO) solvent systems, whereas the peptide forms oligomers in aqueous media. In this study, we utilized ¹H NMR chemical shift changes (Δδ_1H) in aqueous DMSO solution to evaluate the TNM-A/sterol interaction. Because Chol does not dissolve well in this solvent, we used 25-hydroxycholesterol (25-HC) instead, which turned out to interact with membrane-bound TNM-A in a very similar way to that of Chol. We determined the dissociation constant, K_D, by NMR titration experiments and measured the chemical shift changes of TNM-A induced by 25-HC binding in the DMSO solution. Significant changes were observed for several amino acid residues in a certain area of the molecule. The results from the solution NMR experiments, together with previous findings, suggest that the TNM-Chol complex, where the hydrophobic cavity of TNM probably incorporates Chol, becomes less polar by Chol interaction, resulting in a greater accumulation of the peptide in membrane. The deeper penetration of TNM-A into the membrane interior enhances membrane disruption. We also demonstrated that hydroxylated sterols, such as 25-HC that has higher solubility in most NMR solvents than Chol, act as a versatile substitute for sterol and could be used in ¹H NMR-based studies of sterol-binding peptides.     

PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI. During this transamidation reaction, the GPI transamidase forms a carbonyl intermediate with a substrate protein. It was known that the GPI transamidase is a complex containing GAA1 and GPI8. Here, we report two new components of this enzyme: PIG-S and PIG-T. To determine roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates. We also demonstrate that PIG-S and PIG-T form a protein complex with GAA1 and GPI8, and that PIG-T maintains the complex by stabilizing the expression of GAA1 and GPI8. Saccharomyces cerevisiae Gpi16p (YHR188C) and Gpi17p (YDR434W) are orthologues of PIG-T and PIG-S, respectively.     

True interaction mode of porcine pancreatic elastase with FR136706, a potent peptidyl inhibitor

The crystal structure of porcine pancreatic elastase (PPE) complexed with a potent peptidyl inhibitor, FR136706, was solved at 2.2A resolution. FR136706 fits snugly into the extended active site pocket. The benzene moiety of FR136706 induced dramatic movement of the side chain moiety of Arg217 and both moieties formed a pi-pi interaction, which has never been found previously in structures of PPE complexed with inhibitors. This novel interaction mode may lead to design of new types of inhibitors.     

Antibody-based detection of advanced glycation end-products: promises <Emphasis Type="Italic">vs</Emphasis>. limitations

Advanced glycation end-products (AGEs) of the Maillard reaction were originally measured according to their fluorescent and browning properties. A subsequent study with instrumental analyses such as high-performance liquid chromatography and gas chromatography mass spectrometry more clearly demonstrated the involvement of each AGE structure in pathological conditions. Furthermore, immunochemical methods have also been developed to clarify the localization of AGEs in tissues and measurement of AGEs in multiple clinical samples. Although the involvement of AGEs in age-related diseases has progressed due to immunochemical techniques, the relationship between AGE structure and diseases has not been clear because little was known about the epitope structure of each anti-AGE antibody. However, the development of epitope-identified antibodies against AGEs has made it possible to clarify AGE structures involved in diseases. This review discusses not only the usability of anti-AGE antibodies to evaluate AGEs and disease pathology and screen AGE inhibitors, but also describes their usage.     

Phototropins promote plant growth in response to blue light in low light environments

Phototropins (phot1 and phot2) are plant-specific blue light receptors for phototropism, chloroplast movement, leaf expansion, and stomatal opening. All these responses are thought to optimize photosynthesis by helping to capture light energy efficiently, reduce photodamage, and acquire CO2. However, experimental evidence for the promotion of plant growth through phototropins is lacking. Here, we report dramatic phototropin-dependent effects on plant growth. When plants of Arabidopsis thaliana wild type, the phot1 and phot2 mutants, and the phot1 phot2 double mutant were grown under red light, no significant growth differences were observed. However, if a very low intensity of blue light (0.1 micromol m(-2) s(-1)) was superimposed on red light, large increases in fresh weight up to threefold were found in those plants that carried functional PHOT1 genes. When the intensity of blue light was increased to 1 micromol m(-2) s(-1), the growth enhancement was also found in the phot1 single mutant, but not in the double mutant, indicating that phot2 mediated similar responses as phot1 with a lower sensitivity. The effects occurred under low photosynthetically active radiation in particular. The well-known physiological phototropin-mediated responses, including chloroplast movement, stomatal opening, and leaf expansion, in the different lines tested indicated an involvement of these responses in the blue light-induced growth enhancement. We conclude that phototropins promote plant growth by controlling and integrating a variety of responses that optimize photosynthetic performance under low photosynthetically active radiation in the natural environment.